Dear all, I wrote this email to know whether my contrast matrices are conrrect. Many thanks in advance for any advice. I have a difficulty in constructing the contrast matrix of factorial design in limma. In section 8.7 of limma user's guide, a factorial design is discussed. My experimental design resembles the example very much except that my arrays are direct two-color arrays. Therefore the hybridization diagram is a square as follows: WS-MS |..| WU-MU W: wild type M: mutant U: unstimulated S: stimulated (The horizontal and vertical lines stand for direct hybridizations, and the dots in the square have no meanings but to fill the space) What I'm interested in are exactly the same as those in the example. 1. Which genes respond to stimulation in wild type, 2. Which genes respond to stimulation in mutants,and 3. Which genes respond differently in mutants compared to wild type. I constructed the contrast matrices (see below in the code and results) without knowing their validity. Are they right? Part of my code and results are as follows: > sessionInfo() R version 2.6.2 (2008-02-08) i386-pc-mingw32 locale: LC_COLLATE=Chinese_People's Republic of China.936;LC_CTYPE=Chinese_People's Republic of China.936;LC_MONETARY=Chinese_People's Republic of China.936;LC_NUMERIC=C;LC_TIME=Chinese_People's Republic of China.936 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] statmod_1.3.5 limma_2.12.0 > targets Cy5 Cy3 FileName 1 WS WU 1.gpr 2 WU WS 2.gpr 3 MS MU 3.gpr 4 MU MS 4.gpr 5 WS MS 5.gpr 6 MS WS 6.gpr 7 WU MU 7.gpr 8 MU WU 8.gpr > design<-modelMatrix(targets,ref="WU") Found unique target names: MS MU WS WU > design MS MU WS [1,] 0 0 1 [2,] 0 0 -1 [3,] 1 -1 0 [4,] -1 1 0 [5,] -1 0 1 [6,] 1 0 -1 [7,] 0 -1 0 [8,] 0 1 0 > contrast.matrix<-makeContrasts("WS-WU"=WS,"MS-MU"=MS-MU,"(MS-MU )-(WS-WU)"=(MS-MU)-WS,levels=design) > contrast.matrix Contrasts Levels WS-WU MS-MU (MS-MU)-(WS-WU) MS 0 1 1 MU 0 -1 -1 WS 1 0 -1